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Background Blood-based biomarkers are gaining grounds for the detection of Alzheimer’s disease (AD) and related disorders (ADRDs). However, two key obstacles remain: the lack of methods for multi-analyte assessments and the need for biomarkers for related pathophysiological processes like neuroinflammation, vascular, and synaptic dysfunction. A novel proteomic method for pre-selected analytes, based on proximity extension technology, was recently introduced. Referred to as the NULISAseq CNS disease panel, the assay simultaneously measures ~ 120 analytes related to neurodegenerative diseases, including those linked to both core (i.e., tau and amyloid-beta (Aβ)) and non-core AD processes. This study aimed to evaluate the technical and clinical performance of this novel targeted proteomic panel. Methods The NULISAseq CNS disease panel was applied to 176 plasma samples from 113 individuals in the MYHAT-NI cohort of predominantly cognitively normal participants from an economically underserved region in southwestern Pennsylvania, USA. Classical AD biomarkers, including p-tau181, p-tau217, p-tau231, GFAP, NEFL, Aβ40, and Aβ42, were independently measured using Single Molecule Array (Simoa) and correlations and diagnostic performances compared. Aβ pathology, tau pathology, and neurodegeneration (AT(N) statuses) were evaluated with [ 11 C] PiB PET, [ 18 F]AV-1451 PET, and an MRI-based AD-signature composite cortical thickness index, respectively. Linear mixed models were used to examine cross-sectional and Wilcoxon rank sum tests for longitudinal associations between NULISA and neuroimaging-determined AT(N) biomarkers. Results NULISA concurrently measured 116 plasma biomarkers with good technical performance (97.2 ± 13.9% targets gave signals above assay limits of detection), and significant correlation with Simoa assays for the classical biomarkers. Cross-sectionally, p-tau217 was the top hit to identify Aβ pathology, with age, sex, and APOE genotype-adjusted AUC of 0.930 (95%CI: 0.878–0.983). Fourteen markers were significantly decreased in Aβ-PET + participants, including TIMP3, BDNF, MDH1, and several cytokines. Longitudinally, FGF2, IL4, and IL9 exhibited Aβ PET-dependent yearly increases in Aβ-PET + participants. Novel plasma biomarkers with tau PET-dependent longitudinal changes included proteins associated with neuroinflammation, synaptic function, and cerebrovascular integrity, such as CHIT1, CHI3L1, NPTX1, PGF, PDGFRB, and VEGFA; all previously linked to AD but only reliable when measured in cerebrospinal fluid. The autophagosome cargo protein SQSTM1 exhibited significant association with neurodegeneration after adjusting age, sex, and APOE ε4 genotype. Conclusions Together, our results demonstrate the feasibility and potential of immunoassay-based multiplexing to provide a comprehensive view of AD-associated proteomic changes, consistent with the recently revised biological and diagnostic framework. Further validation of the identified inflammation, synaptic, and vascular markers will be important for establishing disease state markers in asymptomatic AD.

Insufficient functional β-cell mass causes diabetes; however, an effective cell replacement therapy for curing diabetes is currently not available. Reprogramming of acinar cells toward functional insulin-producing cells would offer an abundant and autologous source of insulin-producing cells. Our lineage tracing studies along with transcriptomic characterization demonstrate that treatment of adult mice with a small molecule that specifically inhibits kinase activity of focal adhesion kinase results in trans-differentiation of a subset of peri-islet acinar cells into insulin producing β-like cells. The acinar-derived insulin-producing cells infiltrate the pre-existing endocrine islets, partially restore β-cell mass, and significantly improve glucose homeostasis in diabetic mice. These findings provide evidence that inhibition of the kinase activity of focal adhesion kinase can convert acinar cells into insulin-producing cells and could offer a promising strategy for treating diabetes. A cure for diabetes could entail an effective cell replacement therapy through generation of new insulinproducing cells. In this study, we show that inhibition of focal adhesion kinase activity results in transdifferentiation of a subset of peri-islet acinar cells into functional insulin producing β-like cells.

Ayurvedic medicine plants continue to draw attention for the discovery of novel anticancer agents. Withaferin A (WA) is one such small-molecule constituent of the ayurvedic medicine plant Withania somnifera with efficacy against cultured and xenografted human breast cancer cells. However, the mechanism underlying anticancer effect of WA is not fully understood. This study was undertaken to determine the role of Notch signaling in anticancer effects of WA using human breast cancer cells as a model. Notably, Notch signaling is often hyperactive in human breast cancers. Exposure of MDA-MB-231 and MCF-7 human breast cancer cells to pharmacological concentrations of WA resulted in cleavage (activation) of Notch2 as well as Notch4, which was accompanied by transcriptional activation of Notch as evidenced by RBP-Jk, HES-1A/B, and HEY-1 luciferase reporter assays. On the other hand, WA treatment caused a decrease in levels of both transmembrane and cleaved Notch1. The WA-mediated activation of Notch was associated with induction of γ-secretase complex components presenilin1 and/or nicastrin. Inhibition of MDA-MB-231 and MDA-MB-468 cell migration resulting from WA exposure was significantly augmented by knockdown of Notch2 as well as Notch4 protein. Activation of Notch2 was not observed in cells treated with withanone or withanolide A, which are structural analogs of WA. The results of this study indicate that WA treatment activates Notch2 and Notch4, which impede inhibitory effect of WA on breast cancer cell migration.

Alzheimer’s disease (AD), the most common form of dementia, remains challenging to understand and treat despite decades of research and clinical investigation. This might be partly due to a lack of widely available and cost-effective modalities for diagnosis and prognosis. Recently, the blood-based AD biomarker field has seen significant progress driven by technological advances, mainly improved analytical sensitivity and precision of the assays and measurement platforms. Several blood-based biomarkers have shown high potential for accurately detecting AD pathophysiology. As a result, there has been considerable interest in applying these biomarkers for diagnosis and prognosis, as surrogate metrics to investigate the impact of various covariates on AD pathophysiology and to accelerate AD therapeutic trials and monitor treatment effects. However, the lack of standardization of how blood samples and collected, processed, stored analyzed and reported can affect the reproducibility of these biomarker measurements, potentially hindering progress toward their widespread use in clinical and research settings. To help address these issues, we provide fundamental guidelines developed according to recent research findings on the impact of sample handling on blood biomarker measurements. These guidelines cover important considerations including study design, blood collection, blood processing, biobanking, biomarker measurement, and result reporting. Furthermore, the proposed guidelines include best practices for appropriate blood handling procedures for genetic and ribonucleic acid analyses. While we focus on the key blood-based AD biomarkers for the AT(N) criteria (e.g., amyloid-beta [Aβ]40, Aβ42, Aβ42/40 ratio, total-tau, phosphorylated-tau, neurofilament light chain, brain-derived tau and glial fibrillary acidic protein), we anticipate that these guidelines will generally be applicable to other types of blood biomarkers. We also anticipate that these guidelines will assist investigators in planning and executing biomarker research, enabling harmonization of sample handling to improve comparability across studies.

The present study was undertaken to determine the anticancer efficacy of zerumbone (ZER), a sesquiterpene from subtropical ginger, against human breast cancer cells in vitro and in vivo. ZER treatment caused a dose-dependent decrease in viability of MCF-7 and MDA-MB-231 human breast cancer cells in association with G 2 /M phase cell cycle arrest and apoptosis induction. ZER-mediated cell cycle arrest was associated with downregulation of cyclin B1, cyclin-dependent kinase 1, Cdc25C, and Cdc25B. Even though ZER treatment caused stabilization of p53 and induction of PUMA, these proteins were dispensable for ZER-induced cell cycle arrest and/or apoptosis. Exposure of MDA-MB-231 and MCF-7 cells to ZER resulted in downregulation of Bcl-2 but its ectopic expression failed to confer protection against ZER-induced apoptosis. On the other hand, the SV40 immortalized mouse embryonic fibroblasts derived from Bax and Bak double knockout mice were significantly more resistant to ZER-induced apoptosis. ZER-treated MDA-MB-231 and MCF-7 cells exhibited a robust activation of both Bax and Bak. In vivo growth of orthotopic MDA-MB-231 xenografts was significantly retarded by ZER administration in association with apoptosis induction and suppression of cell proliferation (Ki-67 expression). These results indicate that ZER causes G 2 /M phase cell cycle arrest and Bax/Bak-mediated apoptosis in human breast cancer cells, and retards growth of MDA-MB-231 xenografts in vivo.

Chronic pancreatitis is a debilitating disease affecting millions worldwide. These patients suffer from bouts of severe pain that are minimally relieved by pain medications and may necessitate major surgeries with high morbidity and mortality. Previously, we demonstrated that “chemical pancreatectomy,” a pancreatic intraductal infusion of dilute acetic acid solution, ablated the exocrine pancreas while preserving the endocrine pancreas. Notably, chemical pancreatectomy resolved chronic inflammation, alleviated allodynia in the cerulein pancreatitis model, and improved glucose homeostasis. Herein, we extensively tested the feasibility of a chemical pancreatectomy in NHPs and validated our previously published pilot study. We did serial computed tomography (CT) scans of the abdomen and pelvis, analyzed dorsal root ganglia, measured serum enzymes, and performed histological and ultrastructural assessments and pancreatic endocrine function assays. Based on serial CT scans, chemical pancreatectomy led to the loss of pancreatic volume. Immunohistochemistry and transmission electron microscopy demonstrated exocrine pancreatic ablation with endocrine islet preservation. Importantly, chemical pancreatectomy did not increase pro-nociceptive markers in harvested dorsal root ganglia. Also, chemical pancreatectomy improved insulin secretion to supranormal levels in vivo and in vitro. Thus, this study may provide a foundation for translating this procedure to patients with chronic pancreatitis or other conditions requiring a pancreatectomy.

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effect of BITC are not fully understood. Here, we demonstrate that BITC treatment causes FoxO1-mediated autophagic death in cultured human breast cancer cells. The BITC-treated breast cancer cells (MDA-MB-231, MCF-7, MDA-MB-468, BT-474, and BRI-JM04) and MDA-MB-231 xenografts from BITC-treated mice exhibited several features characteristic of autophagy, including appearance of double-membrane vacuoles (transmission electron microscopy) and acidic vesicular organelles (acridine orange staining), cleavage of microtubule-associated protein 1 light chain 3 (LC3), and/or suppression of p62 (p62/SQSTM1 or sequestosome 1) expression. On the other hand, a normal human mammary epithelial cell line (MCF-10A) was resistant to BITC-induced autophagy. BITC-mediated inhibition of MDA-MB-231 and MCF-7 cell viability was partially but statistically significantly attenuated in the presence of autophagy inhibitors 3-methyl adenine and bafilomycin A1. Stable overexpression of Mn-superoxide dismutase, which was fully protective against apoptosis, conferred only partial protection against BITC-induced autophagy. BITC treatment decreased phosphorylation of mTOR and its downstream targets (P70s6k and 4E-BP1) in cultured MDA-MB-231 and MCF-7 cells and MDA-MB-231 xenografts, but activation of mTOR by transient overexpression of its positive regulator Rheb failed to confer protection against BITC-induced autophagy. Autophagy induction by BITC was associated with increased expression and acetylation of FoxO1. Furthermore, autophagy induction and cell growth inhibition resulting from BITC exposure were significantly attenuated by small interfering RNA knockdown of FoxO1. In conclusion, the present study provides novel insights into the molecular circuitry of BITC-induced cell death involving FoxO1-mediated autophagy.

Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive component of edible cruciferous vegetables with in vivo efficacy against prostate cancer in experimental rodents. Cancer chemopreventive response to PEITC is characterized by its ability to inhibit multiple oncogenic signaling pathways, including nuclear factor-κB, Akt, and androgen receptor. The present study demonstrates, for the first time, that PEITC treatment activates Notch signaling in malignant as well as normal human prostate cells. Exposure of human prostate cancer cells (LNCaP, PC-3, and DU145) and a normal human prostate epithelial cell line (PrEC) to PEITC resulted in cleavage (active form) of Notch1 and Notch2, and increased transcriptional activity of Notch. In PC-3 and LNCaP cells, PEITC treatment caused induction of Notch ligands Jagged1 and Jagged2 (PC-3), overexpression of γ-secretase complex components Presenilin1 and Nicastrin (PC-3), nuclear enrichment of cleaved Notch2, and/or up-regulation of Notch1, Notch2, Jagged1, and/or Jagged2 mRNA. PEITC-induced apoptosis in LNCaP and PC-3 cells was significantly attenuated by RNA interference of Notch2, but not by pharmacological inhibition of Notch1. Inhibition of PC-3 and LNCaP cell migration resulting from PEITC exposure was significantly augmented by knockdown of Notch2 protein as well as pharmacological inhibition of Notch1 activation. Nuclear expression of cleaved Notch2 protein was significantly higher in PC-3 xenografts from PEITC-treated mice and dorsolateral prostates from PEITC-fed TRAMP mice compared with respective control. Because Notch signaling is implicated in epithelial-mesenchymal transition and metastasis, the present study suggests that anti-metastatic effect of PEITC may be augmented by a combination regimen involving a Notch inhibitor.

Background Phosphorylated tau proteins accumulate in pathological aggregates which define neurodegenerative tauopathies, including Alzheimer’s disease (AD). Insight into the early stages of tau polymerization/aggregation, including early hyperphosphorylation events, is critical for identification of biomarkers of incipient disease as well as novel therapy targets. Method We analyzed postmortem tissue sections of hippocampus from AD cases and middle frontal gyrus from non‐AD cases with mainly 4R tau isoforms (progressive supranuclear palsy, PSP; corticobasal degeneration, CBD; aging related tau astrogliopathy, ARTAG) or 3R tau (Pick’s disease, PiD). Immunohistochemical and multiple fluorescence methods were used to assess the labeling patterns of antibodies directed to phospo‐epitopes within soluble non‐fibrillar tau core (pSer262), both soluble and fibril core (pSer356), or outside of either core region (pSer202/pThr205, pThr231). Result In sections of hippocampus from AD cases, pSer262 and pSer356 stained punctate/granular tau aggregates in pretangles while pSer202/pThr205 and pThr231 antibodies stained tau in pretangles as well as classic NFT, neuritic component of plaques, and neuropil threads. Both pSer262 and pSer356 signals co‐distributed with pSer202/pThr205 signal in pretangles but were rarely detected in pSer202/pThr205 immunoreactive classic NFT. In sections of middle frontal gyrus from PSP and CBD cases, tufted astrocytes and astrocytic plaques, respectively, were robustly labeled with the pSer202/pThr205 antibody but lacked significant immunoreactivity to pSer262 and pSer356 antibodies. Similarly, thorn shaped astrocytes in ARTAG cases were robustly labeled with the pSer202/pThr205 antibody but were weakly labeled with the pSer262 and pSer356 antibodies. Pick bodies were labeled robustly with the pSer202/pThr205 antibody, moderately with the pSer356 antibody, and weak and sparsely with the pSer262 antibody. Conclusion Antibodies directed against phosphorylated epitopes within core region of soluble and fibrillar tau distinguish hippocampal pretangles in AD and appear to have less affinity for tau pathology forms in non‐AD tauopathies. Thus, they may be novel biomarkers for detecting early stages of neurofibrillary pathology development in AD.

Background Neurofibrillary tangles (NFT), consisting of hyperphosphorylated tau aggregates, are one of the major pathological hallmarks of Alzheimer’s disease (AD). The burden of NFTs correlates with cognitive decline, and in vivo detection of NFT may help predict the clinical progression of AD. Mass spectrometry‐based proteomic analysis of brain regions affected by NFTs holds the potential to unveil the molecular mechanisms underlying tau pathogenesis and uncover novel diagnostic/prognostic biomarkers and therapeutic targets. Method Post‐mortem frozen samples of inferior temporal and middle frontal cortex were collected from 9 cases with different severity of tau pathology assessed by Braak NFT stage. Proteins were fractionated into three fractions using sequential extraction with lysis buffers with different solubility strengths (TBS, Na2CO3, and Urea), trypsin‐digested, and analyzed using label‐free nano‐flow liquid chromatography‐tandem mass spectrometry (nano LC‐MS/MS). Linear mixed models were employed to assess the significance of proteins showing a differential abundance in tissues with Braak NFT stages 0‐III and IV‐VI or displaying Braak stage‐dependent solubility changes. Result A total of 64908 peptides originating from 5475 protein groups were identified through the analysis of 18 brain tissue samples. Differential proteomic analysis revealed several proteins with significantly varied abundance in tissues with different severity of tau pathology. The most significant proteins among these include COL25A1, SNRNP70, MDK, ASPRV1, CLMN, DOCK5, HTRA1, LNPEP, SQSTM1 and Aβ peptides. Additionally, a number of proteins exhibited Braak‐stage dependent solubility change; a significant subset of these proteins, including SNRNP70, SNRPGP15, PRPF6, SNRPE, SNRPD1, SNRPB, and SNRPD3, is involved in the spliceosome‐mediated mRNA splicing. Conclusion Our study reveals numerous proteins exhibiting a significant association with tau pathology. These results align well with published findings that suggest the involvement of alternative splicing in reprogramming gene expression in the pathogenesis of AD. Further validation will be needed to assess their potential as therapeutic targets and/or diagnostic/prognostic biomarkers for AD pathologies.